The phosphoinositides PI4P, PI(4,5)P2, and PI(3,4,5)P3 orchestrate numerous physiological processes occurring at the plasma membrane, including exo/endocytosis, actin assembly, and several growth factor signaling pathways, and dysregulation of the metabolism of these lipids is causative of many diseases, including diabetes, numerous cancers, and several neurological disorders. The first committed step in the biosynthesis of these phosphoinositides is catalyzed by the PI 4-kinase Type III? (PI4KIII?). Despite the fundamental role that this enzyme plays in the phosphoinositide metabolic network, surprisingly little is known about its regulation. To understand how nature controls the subcellular localization and activity of PI4KIII? and hence regulates the phosphoinositide pools under its control, I have in my preliminary work characterized two PI4KIII? regulatory proteins and begun to study their molecular properties and physiological functions. I showed that the palmitoylated, peripheral membrane protein EFR3/Rolling blackout cooperates with a soluble tetratricopeptide adaptor, TTC7, to recruit PI4KIII? to the plasma membrane, its site of action. Recently, I identified a third potential PI4KIII? regulator, hyccin, a protein of unknown function implicated in a brain white matter disease termed hypomyelination and congenital cataracts (HCC). The overarching hypothesis guiding this work is that EFR3, TTC7, and hyccin form the core of a PI4KIII? protein-protein interaction network that controls PI4P synthesis at the plasma membrane. Aim 1 describes studies to elucidate fundamental cellular functions of EFR3 and test the hypothesis that EFR3 is a master regulator of phosphoinositide metabolism at the neuronal synapse. Aim 2 outlines biochemical and chemical biology experiments to define the principles governing the assembly of the PI4KIII?/TTC7/EFR3 complex and its spatiotemporal regulation at the plasma membrane. Aim 3 describes studies to probe the physical and functional connection between hyccin and PI4KIII?, TTC7, and EFR3. In sum, these multidisciplinary studies will elucidate fundamental principles that regulate the PI4P synthetic machinery at the plasma membrane. More broadly, the approaches I develop and the principles that I elucidate will be applicable to the long-term goal of understanding how regulation of PI4P synthesis connects to the broader metabolic network (e.g., via coupling to downstream enzymes that generate PI(4,5)P2, PI(3,4,5)P3, IP3, and diacylglycerol). As well, my proposed studies to connect hyccin function to PI4P metabolism represent a first step toward the long-term goal of understanding the mechanisms of pathogenesis of HCC, which may shed light on potential therapies for this and other leukodystrophies.